I’ve been trying to work out the ferrozine method for measuring ferrous iron and have been unsuccessful at getting a color reaction in a reasonable amount of time. I started out using a protocol from Van Bodegom et al 2003 (0.1 ml sample plus 0.9 ml ferrozine/HEPES buffer, pH 4.0) but ended up with a pH well below 4 after adding the ferrozine/buffer and my standards. I did, however, end up getting color development but only after a good 45 minutes.
I tried to remedy the reaction by raising the ferrozine/HEPES buffer to pH 7.0 and also remaking standards in 0.1 M HCl instead of 0.5 M HCl. I also tried increasing the amount of ferrozine (5 ml to 0.1 ml of sample). Again, faint color development only after 30 minutes. I have been working with standards between the range of 5 mM and 0.5 mM Fe2+. Most pubs state they measure immediately after shaking the concoction for 10 seconds. A few will wait 5 or 10 minutes for color development. All of my reagents are new. I’ve checked and rechecked concentrations and everything looks good. This assay should be easy as pie. Any tips would be appreciated. Feel free to e-mail me privately with suggestions.
